Isomeric N-glycans often vastly differ in their biological activities, hence the need for methods that allow resolving and structurally characterizing them in biological material. Here, we... Show moreIsomeric N-glycans often vastly differ in their biological activities, hence the need for methods that allow resolving and structurally characterizing them in biological material. Here, we established a zero flow approach using capillary electrophoresis in combination with (tandem) mass spectrometry to allow structural characterization of isomeric N-glycans at high sensitivity. Additionally, diagnostic fragment ion ratios were identified, indicative for the antenna carrying specifically linked sialic acids. In total, 208 N-glycans were characterized in human plasma, with 57 compositions showing multiple isomers. Show less
The desolvation and ionization process of analytes can significantly be improved by enriching the nebulizing gas with a dopant (dopant enriched nitrogen (DEN) gas) in the electrospray source.... Show moreThe desolvation and ionization process of analytes can significantly be improved by enriching the nebulizing gas with a dopant (dopant enriched nitrogen (DEN) gas) in the electrospray source. However, for the analysis of released glycans in negative ion mode, the usage of DEN gas remains largely unexplored. For this purpose, we investigated the effect of different polar protic solvents (methanol, ethanol, and isopropanol) as well as using solely the nebulizing gas or ambient air on the ionization and charge state distribution of released N- and O-glycans. Compared to the standard acetonitrile enriched nitrogen gas, isopropanol showed the highest increase in regards to peak areas. Moreover, it showed large benefits for the identification of glycan structures at high sensitivity as the increased precursor intensities subsequently resulted in higher intensities in tandem MS mode. While similar effects are noted for both neutral and sialylated species, the most significant effect was observed for early eluting glycans where very low acetonitrile concentrations were present in the eluent. The best results in terms of S/N ratios were obtained with methanol, with less effect on the MS/MS signal enhancement. Therefore, the use of this dopant would be particularly beneficial for high sensitivity MS-mode applications. In conclusion, isopropanol enriched DEN gas greatly improves the detection of both N-and O-glycan species and their tandem mass spectra, particularly for the early eluting species whose ionization in the absence of DEN gas is hindered by low organic concentrations. Show less
Background: Effector functions of IgG Abs are regulated by their Fc N-glycosylation pattern. IgG Fc glycans that lack galactose and terminal sialic acid residues correlate with the severity of... Show moreBackground: Effector functions of IgG Abs are regulated by their Fc N-glycosylation pattern. IgG Fc glycans that lack galactose and terminal sialic acid residues correlate with the severity of inflammatory (auto)immune disorders and have also been linked to protection against viral infection and discussed in the context of vaccine-induced protection. In contrast, sialylated IgG Abs have shown immunosuppressive effects.Objective: We sought to investigate IgG glycosylation programming during the germinal center (GC) reaction following immunization of mice with a foreign protein antigen and different adjuvants.Methods: Mice were analyzed for GC T-cell, B-cell, and plasma cell responses, as well as for antigen-specific serum IgG subclass titers and Fc glycosylation patterns.Results: Different adjuvants induce distinct IgG(+) GC B-cell responses with specific transcriptomes and expression levels of the alpha 2,6-sialyltransferase responsible for IgG sialylation that correspond to distinct serum IgG Fc glycosylation patterns. Low IgG Fc sialylation programming in GC B cells was overall highly dependent on the Foxp3(-) follicular helper T (TFH) cell-inducing cytokine IL-6, here in particular induced by water-inoil adjuvants and Mycobacterium tuberculosis. Furthermore, low IgG Fc sialylation programming was dependent on adjuvants that induced IL-27 receptor-dependent IFN-gamma(+) TFH1 cells, IL-6/IL-23-dependent IL-17A(+) T-FH17 cells, and high ratios of TFH cells to Foxp31 follicular regulatory T cells. Here, the 2 latter were dependent on M tuberculosis and its cord factor.Conclusion: This study's findings regarding adjuvant-dependent GC responses and IgG glycosylation programming may aid in the development of novel vaccination strategies to induce IgG Abs with both high affinity and defined Fc glycosylation patterns in the GC. Show less
Lageveen-Kammeijer, G.S.M.; Haan, N. de; Mohaupt, P.; Wagt, S.; Filius, M.; Nouta, J.; ... ; Wuhrer, M. 2019
